模擬和光譜相互驗(yàn)證2'-OH-BDE-28對(duì)HSA構(gòu)象的影響

伍智蔚，易忠勝,*，董露，楊露露，楊霧，張愛(ài)茜

1.廣西高校食品安全與檢測(cè)重點(diǎn)實(shí)驗(yàn)室，巖溶地區(qū)水污染控制與用水安全保障協(xié)同創(chuàng)新中心，桂林理工大學(xué)化學(xué)與生物工程學(xué)院，桂林541004

2.中國(guó)科學(xué)院生態(tài)環(huán)境研究中心環(huán)境化學(xué)與生態(tài)毒理學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室，北京100085

模擬和光譜相互驗(yàn)證2'-OH-BDE-28對(duì)HSA構(gòu)象的影響

伍智蔚1，易忠勝1,*，董露1，楊露露1，楊霧1，張愛(ài)茜2

1.廣西高校食品安全與檢測(cè)重點(diǎn)實(shí)驗(yàn)室，巖溶地區(qū)水污染控制與用水安全保障協(xié)同創(chuàng)新中心，桂林理工大學(xué)化學(xué)與生物工程學(xué)院，桂林541004

2.中國(guó)科學(xué)院生態(tài)環(huán)境研究中心環(huán)境化學(xué)與生態(tài)毒理學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室，北京100085

利用分子對(duì)接、分子動(dòng)力學(xué)模擬(molecular dynamics simulation,MD)和光譜法研究2'-羥基-2,4,4'-三溴二苯醚(2'-OHBDE-28)與人血清白蛋白(HSA)的作用機(jī)制。MD模擬研究表明2'-OH-BDE-28誘導(dǎo)HSA的內(nèi)部疏水性增強(qiáng),結(jié)構(gòu)松散膨脹,致使其二級(jí)結(jié)構(gòu)發(fā)生改變；圓二色光譜實(shí)驗(yàn)與MD模擬結(jié)果相吻合,驗(yàn)證2'-OH-BDE-28可誘導(dǎo)HSA的構(gòu)象變化。熒光光譜實(shí)驗(yàn)表明,2'-OH-BDE-28能通過(guò)靜態(tài)猝滅和非輻射能量轉(zhuǎn)移機(jī)制引起HSA熒光猝滅。分子對(duì)接推斷2'-OH-BDE-28以氫鍵和疏水作用力鍵合在HSA的位點(diǎn)I處；熱力學(xué)分析和競(jìng)爭(zhēng)實(shí)驗(yàn)結(jié)果一致驗(yàn)證分子對(duì)接結(jié)果。本文將計(jì)算模擬和光譜實(shí)驗(yàn)相結(jié)合,從模擬和實(shí)驗(yàn)2個(gè)角度共同探討2'-OH-BDE-28與HSA的作用機(jī)制,結(jié)果高度吻合。

2'-羥基-2,4,4'-三溴二苯醚；人血清白蛋白；分子動(dòng)力學(xué)模擬；光譜法

環(huán)境污染物多溴二苯醚(polybrominated diphenyl ethers,PBDEs)作為溴代阻燃劑因其難降解,生物毒性大且蓄積性等特點(diǎn)[1-2],已經(jīng)引起人們的廣泛關(guān)注。自20世紀(jì)60年代以來(lái),由于長(zhǎng)期大量使用PBDEs使得環(huán)境介質(zhì)中PBDEs存在含量逐年遞增,覆蓋范圍擴(kuò)散得較廣[3-5]。近年來(lái),研究工作者在水體、大氣、農(nóng)副產(chǎn)品甚至人體內(nèi)不但陸續(xù)檢測(cè)出多溴二苯醚還檢測(cè)出其典型的衍生物-羥基化多溴二苯醚(OH-PBDEs)[6-8],其化學(xué)結(jié)構(gòu)與母體多溴二苯醚相似,除了具備母體PBDEs的一些毒性特征外,OHPBDEs還具有一些特殊的毒理特征[7,9-10]。同時(shí), OH-PBDEs生物毒性在某些方面要大于其母體[11],它對(duì)環(huán)境和人類的危害更是不可忽視。目前,國(guó)內(nèi)外研究者們對(duì)于OH-PBDEs的研究還處于起步階段,其轉(zhuǎn)化行為、毒理效應(yīng)、作用機(jī)制仍不甚清晰。

人血清白蛋白(HSA)是人血液最為豐富的蛋白質(zhì),承載著物質(zhì)運(yùn)輸與轉(zhuǎn)載,同時(shí)還維持血液膠體的滲透壓。HSA也是具有顯要生理功能的蛋白質(zhì),它可以與進(jìn)入體內(nèi)的外源性物質(zhì)進(jìn)行快速、可逆的結(jié)合[12],進(jìn)而發(fā)揮外源性物質(zhì)的生物學(xué)作用,因此研究具有藥理活性的小分子與HSA相互作用,對(duì)于了解藥物小分子的藥理、毒理機(jī)制具有十分重要的意義。

本文以 2'-羥基-2,4,4'-三溴二苯醚(2'-OHBDE-28)為例,利用計(jì)算模擬和光譜法相結(jié)合研究2 '-OH-BDE-28與HSA的作用機(jī)制,探討其結(jié)合情況:包括2'-OH-BDE-28與HSA結(jié)合的分子機(jī)制(結(jié)合構(gòu)象和結(jié)合位點(diǎn))、熒光猝滅機(jī)制以及2'-OHBDE-28對(duì)HSA穩(wěn)定性和二級(jí)結(jié)構(gòu)的影響。

1 材料與方法(Materials and methods)

1.1 儀器與試劑

RF-5301PC型熒光分光光度計(jì)(日本島津)；TU-1901型雙光束紫外可見(jiàn)分光光度計(jì)(北京普析)；J-810型圓二色光譜儀(日本分光株式會(huì)社)；PHS-3C型酸度計(jì)(上海雷磁)。

2'-OH-BDE-28(purity≥99%,購(gòu)于德國(guó)DR公司):用pH 7.4的Tris-HCl緩沖溶液(含NaCl 0.15 mol·L-1)配制1.0×10-6mol·L-1儲(chǔ)備液。HSA(purity≥97%,購(gòu)于美國(guó)sigma公司):配制1.0×10-5mol·L-1儲(chǔ)備液,用Tris-HCl緩沖溶液定容,4℃以下避光儲(chǔ)存?zhèn)溆?；其他試劑均為分析?實(shí)驗(yàn)用水均為二次蒸餾水。

本文所有的計(jì)算工作均在DELL服務(wù)器的RedHat Linux 6.4系統(tǒng)上完成。使用Sybyl x 1.1軟件進(jìn)行分子對(duì)接和GROMACS 5.0軟件進(jìn)行分子動(dòng)力學(xué)模擬。所用的HSA晶體結(jié)構(gòu)(代碼為1O9X, 1O9X由去掉波動(dòng)區(qū)域較大C、N-末端氨基酸殘基的579個(gè)氨基酸構(gòu)成,殘基區(qū)域?yàn)?～582,結(jié)構(gòu)相對(duì)完整,在位點(diǎn)I和位點(diǎn)II處均含有對(duì)應(yīng)配體,便于計(jì)算模擬)從 Brookhaven蛋白質(zhì)數(shù)據(jù)庫(kù)(http://www. rcsb.org/pdb)獲得。

1.2 實(shí)驗(yàn)方法

分子對(duì)接:運(yùn)用ChemBioDraw Ultra 12.0軟件構(gòu)建2'-OH-BDE-28結(jié)構(gòu),選擇MMFF94力場(chǎng)進(jìn)行能量最小化優(yōu)化結(jié)構(gòu)；HSA選擇AMBER7 FF99力場(chǎng)的最陡下降法-共軛梯度法能量最小化；采用CH4,C=O,N-H為分子探針進(jìn)行柔性對(duì)接。

分子動(dòng)力學(xué)模擬(MD):利用GROMACS 5.0程序分別進(jìn)行10 ns的未配位HSA和2'-OH-BDE-28-HSA復(fù)合物的MD模擬。采用GROMOS96 43A1力場(chǎng),通過(guò)最陡下降法進(jìn)行初始結(jié)構(gòu)的能量最小化,并采用正則系統(tǒng)(NVT)和等溫等壓系統(tǒng)(NPT)平衡體系進(jìn)行10 ns分子動(dòng)力學(xué)模擬,最后用VMD軟件和LigPlus軟件進(jìn)行分子圖形展示和結(jié)果分析。

光譜法:在10 mL的比色管中準(zhǔn)確移入1 mL 1.0×10-5mol·L-1HSA溶液,依次加入一定量的1.0×10-6mol·L-12'-OH-BDE-28溶液,用pH 7.4 Tris-HCl緩沖溶液定容至刻度線,搖勻,在設(shè)定溫度291 K、298 K、310 K下恒溫反應(yīng)5 min,掃描激發(fā)波長(zhǎng)為285 nm下的發(fā)射光譜,儀器的激發(fā)和發(fā)射熒光狹縫均為5 nm。以Tris-HCl緩沖液為參比,測(cè)定其相應(yīng)的圓二色光譜和紫外光譜。

2 結(jié)果(Results)

2.1 HSA與2'-OH-BDE-28復(fù)合物的結(jié)構(gòu)特征

首先要指明的是本文中HSA體系代表沒(méi)有添加2'-OH-BDE-28的空白體系,2'-OH-BDE-28-HSA體系代表HSA與2'-OH-BDE-28結(jié)合復(fù)合物的體系。

圖1 HSA體系和2'-OH-BDE-28-HSA體系的動(dòng)力學(xué)模擬結(jié)果注:(a)蛋白質(zhì)骨架的均方根偏差值(RMSD)與時(shí)間的關(guān)系；(b)蛋白質(zhì)的均方根波動(dòng)值(RMSF)與殘基的關(guān)系；(c)蛋白質(zhì)的回旋半徑；(d)兩個(gè)體系中親水和疏水的溶劑可及表面積平均值。Fig.1 The results of HSA(black)and 2'-OH-BDE-28-HSA complex(red)during 10 ns MD simulationNote:(a)Time dependence of RMSD values for protein backbone;(b)The RMSF values of protein were against residue numbers; (c)Time evolution of the protein of Gyration;(d)The average of solvent accessible surface areas for hydrophobic and hydrophilic in two systems.

均方根偏差(RMSD)是用來(lái)衡量體系波動(dòng)性的重要參數(shù),而基于殘基分析的均方根波動(dòng)(RMSF)則可以用來(lái)觀察蛋白質(zhì)殘基柔性的變化情況[13]。圖1 a為模擬10 ns HSA體系和2'-OH-BDE-28-HSA體系基于骨架的RMSD值的變化圖。2'-OH-BDE-28-HSA體系整個(gè)模擬過(guò)程中RMSD值波動(dòng)幅度小于HSA體系骨架的波動(dòng)值,這表明了2'-OH-BDE-28與HSA形成穩(wěn)定的復(fù)合物使體系趨于穩(wěn)定。該結(jié)果與前人報(bào)道HSA與其他小分子的RMSD分析結(jié)果類似,例如席夫堿(Schiff base)[14]。圖1 b為10 ns HSA體系和2'-OH-BDE-28-HSA體系氨基酸殘基相對(duì)于平衡位置的波動(dòng)情況。2'-OH-BDE-28-HSA與HSA體系的RMSF變化相似,波動(dòng)較小,這反映了在MD模擬過(guò)程中HSA殘基沒(méi)有較大的位置變動(dòng),整個(gè)HSA的氨基酸柔性發(fā)生微弱的改變。但在氨基酸殘基196～297(位點(diǎn)I)處(見(jiàn)圖1 b放大圖),2 '-OH-BDE-28-HSA體系RMSF值略高于HSA體系,這說(shuō)明2'-OH-BDE-28與HSA的相互作用對(duì)此氨基酸殘基柔性影響較大,致使其周圍環(huán)境發(fā)生改變,進(jìn)而影響其二級(jí)結(jié)構(gòu)(見(jiàn)表1的分析結(jié)果)。

回旋半徑(Rg)值是可以用來(lái)衡量HSA結(jié)構(gòu)的緊密程度[15]。圖1 c為10 ns的HSA體系和2'-OHBDE-28-HSA體系的Rg值變化圖。2'-OH-BDE-28-HSA體系的Rg值大于HSA體系,這表明2'-OHBDE-28與HSA結(jié)合后,使氨基酸殘基間微環(huán)境改變,殘基間相互作用(例如HSA與2'-OH-BDE-28之間產(chǎn)生了氫鍵作用和疏水相互作用,見(jiàn)對(duì)接模型結(jié)果)使HSA體系變得膨脹,導(dǎo)致其構(gòu)象發(fā)生改變,這個(gè)變化結(jié)果與圓二色光譜實(shí)驗(yàn)結(jié)果相吻合。同時(shí),其他研究工作者的研究成果[16-17]也與本文的實(shí)驗(yàn)結(jié)果相似:在MD模擬的過(guò)程中HSA的構(gòu)象發(fā)生了改變。

2'-OH-BDE-28使HSA體系氨基酸殘基的可及表面積(solvent accessible surface areas,SASA)發(fā)生改變,進(jìn)而影響其體系的疏水性(見(jiàn)圖1 d)。2'-OHBDE-28能使HSA體系的疏水性殘基的SASA由251.44 nm2變化為321.40 nm2,增加了27.82%,而親水性殘基的SASA則由700.34 nm2變化為614.46 nm2,減少了12.26%。因此得出,2'-OH-BDE-28與HSA結(jié)合使HSA體系的疏水性增強(qiáng),疏水作用力驅(qū)使HSA體系變得膨脹、松散與2'-OH-BDE-28結(jié)合得更穩(wěn)定。這也表明2'-OH-BDE-28與HSA的結(jié)合過(guò)程是一種柔性的結(jié)合過(guò)程。

2.2 HSA二級(jí)結(jié)構(gòu)的影響

根據(jù)文獻(xiàn)[18],蛋白質(zhì)的紫外區(qū)(190～240 nm)圓二色性光譜可用來(lái)預(yù)估其二級(jí)結(jié)構(gòu)中α-螺旋含量的變化情況。圖2 a為2'-OH-BDE-28與HSA相互作用的CD光譜。208 nm和222 nm處出現(xiàn)2個(gè)負(fù)槽,這是典型的α-螺旋結(jié)構(gòu)CD光譜信號(hào)。2'-OHBDE-28加入后HSA在208 nm和222 nm處的負(fù)光譜信號(hào)有所減少,但譜線形狀沒(méi)有發(fā)生明顯的改變。利用CD Pro軟件對(duì)數(shù)據(jù)分析(見(jiàn)表1)得出:2'-OHBDE-28使 HSA的 α-螺旋含量從 42.4%降至41.2%,這說(shuō)明2'-OH-BDE-28進(jìn)入HSA后,與氨基酸殘基的存在力的相互作用,使α-螺旋含量降低, β-折疊含量增加,驅(qū)使內(nèi)部結(jié)構(gòu)松散、膨脹,進(jìn)而對(duì)HSA的二級(jí)結(jié)構(gòu)產(chǎn)生一定影響[19]。同時(shí)數(shù)據(jù)轉(zhuǎn)角、無(wú)規(guī)則卷曲等含量也發(fā)生一定的變化,但α-螺旋向β-折疊轉(zhuǎn)變起主導(dǎo)地位。此外,使用do_dssp程序分析動(dòng)力學(xué)模擬數(shù)據(jù)發(fā)現(xiàn):當(dāng)2'-OH-BDE-28與HSA形成復(fù)合物時(shí),α-螺旋的含量下降(73.0%～72.0%),并化為轉(zhuǎn)角結(jié)構(gòu)(6.0%～7.0%,見(jiàn)表1)。該結(jié)果與CD光譜的分析結(jié)果相似,說(shuō)明2'-OH-BDE-28與HSA結(jié)合時(shí)是以一種柔性的方式進(jìn)行結(jié)合。通過(guò)改變HSA的二級(jí)結(jié)構(gòu)使生成的2'-OH-BDE-28-HSA復(fù)合物更穩(wěn)定。

2.3 2'-OH-BDE-28對(duì)HSA的熒光猝滅機(jī)制

蛋白質(zhì)的熒光猝滅因機(jī)理不同主要分為動(dòng)態(tài)猝滅、靜態(tài)猝滅和非輻射能量轉(zhuǎn)移等。為了進(jìn)一步判斷2'-OH-BDE-28與HSA相互作用的熒光猝滅機(jī)制,通常選定Stern-Volmer方程進(jìn)行數(shù)據(jù)分析,其方程如下:

選定291 K、298 K、310 K三個(gè)溫度進(jìn)行熒光實(shí)驗(yàn),2'-OH-BDE-28加入后,HSA的熒光強(qiáng)度呈現(xiàn)出有規(guī)律的猝滅,并發(fā)生了微弱的藍(lán)移,這與MD模擬中疏水性增強(qiáng)相一致。而3個(gè)溫度下的猝滅速率常數(shù)Ksv的結(jié)果分析(見(jiàn)圖2 b),Ksv隨著溫度升高而降低,且2'-OH-BDE-28對(duì)HSA的Kq值也遠(yuǎn)大于各類猝滅劑對(duì)生物大分子最大動(dòng)態(tài)的猝滅常數(shù)2.0× 1010L·mol-1·S-1。因此,2'-OH-BDE-28猝滅HSA熒光強(qiáng)度的猝滅機(jī)制是靜態(tài)猝滅。這是由于2'-OH-BDE-28進(jìn)入HSA的空腔后,與基態(tài)的HSA發(fā)生了配合反應(yīng),兩者之間相結(jié)合形成復(fù)合物,使HSA內(nèi)部的疏水性增強(qiáng),引起了HSA構(gòu)象的變化,最終導(dǎo)致HSA的熒光強(qiáng)度發(fā)生猝滅。

表1 HSA二級(jí)結(jié)構(gòu)的估計(jì)結(jié)果Table 1 Fractions of secondary structures of HSA by CDPro anddo_dsspprogram

圖2 HSA與2'-OH-BDE-28的光譜實(shí)驗(yàn)結(jié)果注:(a)HSA和2'-OH-BDE-28-HSA體系的圓二色光譜(a-b,cHSA:c2'-OH-BDE-28=1:0,5:1)；(b)Stern-Vomer曲線(T=291 K、298 K、310 K時(shí))；(c)HSA的熒光光譜和2'-OH-BDE-28的紫外吸收光譜重疊圖(T=298 K,cHSA=c2'-OH-BDE-28=1.0×10-7mol·L-1)；(d)華法林競(jìng)爭(zhēng)實(shí)驗(yàn)熒光圖(cHSA=1.0×10-6mol·L-1)。Fig.2 The details of multispectral investigation between HSA and 2'-OH-BDE-28Note:(a)CD spectra of HSA and 2'-OH-BDE-28-HSA system.a-b,cHSA:c2'-OH-BDE-28=1:0,5:1;(b)Stern-Vomer curves at three different temperatures; (c)spectral overlap between the fluorescence emission spectrum of HSA and absorption spectra of 2'-OH-BDE-28 at 298 K, cHSA=c2'-OH-BDE-28=1.0×10-7mol·L-1;(d)fluoresence competitive test by warfarin,cHSA=1.0×10-6mol·L-1.

根據(jù)F?rster偶極-偶極非輻射能量轉(zhuǎn)移理論可得到2'-OH-BDE-28與HSA能量轉(zhuǎn)移信息。選定濃度1:1的2'-OH-BDE-28和HSA,獲得2'-OHBDE-28紫外吸收光譜和HSA熒光光譜的重疊圖譜(見(jiàn)圖2 c)。經(jīng)數(shù)據(jù)分析得出重疊積分J=4.90× 10-14cm3·L·mol-1,臨界距離R0=3.33 nm,能量轉(zhuǎn)移效率E=0.40和2'-OH-BDE-28與HSA中的色氨酸殘基的距離r=3.57 nm＜7 nm且滿足0.5R＜r＜1.5R,符合F?rster偶極-偶極非輻射能量轉(zhuǎn)移的條件[20]。與之前OH-PBDEs與HSA的研究類似[27],說(shuō)明2'-OH-BDE-28與HSA結(jié)合發(fā)生能量轉(zhuǎn)移機(jī)制并引起其熒光猝滅,且能量轉(zhuǎn)移的效率較高。故2'-OH-BDE-28是通過(guò)靜態(tài)猝滅和能量轉(zhuǎn)移2種機(jī)制導(dǎo)致HSA的熒光猝滅。

2.4 2'-OH-BDE-28與HSA作用力類型

研究表明[20],分子對(duì)接可以探討藥物小分子與HSA結(jié)合情況。而HSA的IIA和IIIA是大部分藥物分子的活性位點(diǎn),也稱為位點(diǎn)I(華法林位點(diǎn))和位點(diǎn)II(布洛芬位點(diǎn))。運(yùn)用Sybyl x 1.1軟件,將2'-OH-BDE-28分別與HSA的位點(diǎn)I、位點(diǎn)II進(jìn)行分子對(duì)接,并選取較高得分位點(diǎn)I進(jìn)行分析。圖3為2'-OH-BDE-28與HSA對(duì)接作用圖。2'-OH-BDE-28結(jié)合在HSA位點(diǎn)I處,其5 ?范圍內(nèi)氨基酸殘基(見(jiàn)圖3 a)。其中HSA中ALA291、LEU238、HIS242、ARG257在2'-OH-BDE-28周圍形成“半包裹”的疏水性界面,同時(shí)LYS199通過(guò)2個(gè)氫鍵與其穩(wěn)定鍵合(見(jiàn)圖3 b),氫鍵和疏水作用力對(duì)2'-OH-BDE-28-HSA的穩(wěn)定性起著重要作用。對(duì)接結(jié)果還得出,298 K下2'-OH-BDE-28與HSA的結(jié)合自由能為-15.27 kJ·mol－1。此外,測(cè)量出 2'-OH-BDE-28與TRP214的距離為4.13 ?＜7 nm(見(jiàn)圖3a),完全符合能量轉(zhuǎn)移條件。

圖3 2'-OH-BDE-28-HSA模型分子對(duì)接結(jié)果Fig.3 The results of molecular dockingNote:(a)residues around 6 ? of the 2'-OH-BDE-28 in the active pocket of HSA;(b)two-dimensional plot of hydrophobic effect for the 2'-OH-BDE-28-HSA complex.

根據(jù)文獻(xiàn)[21],分析熒光猝滅實(shí)驗(yàn)數(shù)據(jù)可知2'-OH-BDE-28與HSA相互作用時(shí)的表觀結(jié)合常數(shù)Ka、結(jié)合位點(diǎn)數(shù)n和熱力學(xué)參數(shù)(見(jiàn)表2)。3個(gè)溫度下其結(jié)合位點(diǎn)數(shù)n均約為1,表明在2'-OH-BDE-28與HSA作用時(shí)只有一個(gè)結(jié)合位點(diǎn)。2'-OH-BDE-28與HSA的表觀結(jié)合常數(shù)Ka較大,在106數(shù)量級(jí)以上,說(shuō)明兩者的結(jié)合較牢固,在其與蛋白質(zhì)發(fā)生相互作用結(jié)合后可被蛋白質(zhì)儲(chǔ)存和轉(zhuǎn)運(yùn)。而熱力學(xué)參數(shù)ΔG＜0、ΔH＜0、△S＜0,可以推斷,2'-OH-BDE-28和HSA之間的相互作用是一種自發(fā)的放熱過(guò)程,且其主要驅(qū)動(dòng)力為氫鍵和疏水作用力[21],這也與分子對(duì)接結(jié)果完全吻合。

表2 2'-OH-BDE-28-HSA體系的結(jié)合常數(shù)、位點(diǎn)數(shù)和熱力學(xué)參數(shù)Table 2 Binding constants(Ka),number of binding sites(n)and thermodynamic parameters of 2’-OH-BDE-28-HSA system at different temperatures

競(jìng)爭(zhēng)試驗(yàn)可有效、快捷地判斷2'-OH-BDE-28與HSA的結(jié)合區(qū)域和結(jié)合位點(diǎn)。通常采用布洛芬(ibuprofen)、華法林(warfarin)等熒光探針對(duì)蛋白質(zhì)不同區(qū)域進(jìn)行特異性結(jié)合,然后根據(jù)小分子在HSA結(jié)合位置上的置換作用確定其結(jié)合位點(diǎn),結(jié)果見(jiàn)圖2 d。同時(shí),競(jìng)爭(zhēng)試驗(yàn)的結(jié)合常數(shù)可以通過(guò)修正的Stern-Volmer方程得出

加入Ibuprofen探針后2'-OH-BDE-28與HSA的結(jié)合常數(shù)Ka為9.23×106L·mol-1與未加探針之前的結(jié)合常數(shù)9.90×106L·mol-1很接近,而加入Warfarin探針后2'-OH-BDE-28與HSA的結(jié)合常數(shù)Ka為3.67×106L·mol-1明顯比未加探針之前的9.90×106L·mol-1小很多。這表明2'-OH-BDE-28與HSA的結(jié)合位點(diǎn)是Warfarin位點(diǎn)。

3 討論(Discussion)

目前,用于研究小分子與生物大分子相互作用的技術(shù)有許多。包括光譜法[22]、電化學(xué)方法[23]、核磁共振分析法[24]、質(zhì)譜法[25]等。近年來(lái),計(jì)算模擬技術(shù)在研究相互作用領(lǐng)域有了很大的發(fā)展[26-27]。Rehman等[28]通過(guò)分子對(duì)接技術(shù)直觀的展示了HSA在HSA的IIIA子域穩(wěn)定的與Leu387、Ile388、Asn391、Cys-392、Phe-403、Leu-407、Arg-410、Lys-414、Leu-430等氨基發(fā)生相互作用,并且分子對(duì)接的結(jié)果與熱力學(xué)分析結(jié)果高度吻合。然而,每種方法各有其獨(dú)特之處,也有其局限性。例如光譜法雖然可以測(cè)定小分子與生物大分子形成復(fù)合物前后結(jié)構(gòu)變化的信息,但各種光譜學(xué)方法應(yīng)用范圍有限,需多種光譜學(xué)方法并用[29]。電化學(xué)方法難以測(cè)定一些難電離的小分子例如本文中的OH-PBDEs。而核磁共振分析法所用樣品量較大對(duì)于研究超大型生物大分子具有局限性。因此要打破這些局限,一方面需要多種分析檢測(cè)方法的綜合應(yīng)用和不同學(xué)科之間的協(xié)作。Gao等[30]結(jié)合質(zhì)子核磁共振(H NMR)、紫外可見(jiàn)光譜、熒光光譜、圓二色光譜法研究改性環(huán)糊精與牛血清白蛋白的作用機(jī)制。這項(xiàng)研究結(jié)合了多種手段的優(yōu)點(diǎn)多方面解讀其作用機(jī)制:包括作用前后質(zhì)子的轉(zhuǎn)移、熒光猝滅機(jī)制、α-螺旋的變化以及親和力的強(qiáng)弱等。另一方面,也依賴于實(shí)驗(yàn)方法合理安排以及各種分析測(cè)試技術(shù)的不斷發(fā)展完善。Ma等[25]通過(guò)合理的優(yōu)化設(shè)置利用電噴霧電離質(zhì)譜(ESI/MS)研究了牛血清白蛋白(BSA)與24種不同溴和羥基取代位置的OH-PBDEs同系物的共價(jià)絡(luò)合相互作用。不僅能測(cè)定OH-PBDEs同系物對(duì)各個(gè)BSA的結(jié)合力大小還能推斷BSA的構(gòu)象變化。但目前對(duì)于OH-PBDEs與HSA相互作用的報(bào)道甚少[31]。

本研究也是采用了多種手段結(jié)合研究的方法研究2'-OH-BDE-28與HSA的毒理作用。結(jié)果表明2 '-OH-BDE-28通過(guò)靜態(tài)猝滅和能量轉(zhuǎn)移機(jī)制猝滅HSA的熒光強(qiáng)度,并且誘導(dǎo)HSA的二級(jí)結(jié)構(gòu)發(fā)生變化,其中α-螺旋改變起主導(dǎo)地位。此外,熱力學(xué)分析表明,氫鍵和疏水作用力是維持2'-OH-BDE-28與HSA穩(wěn)定結(jié)合的作用力,這與分子對(duì)接的結(jié)果完全吻合。最后,競(jìng)爭(zhēng)實(shí)驗(yàn)和分子對(duì)接結(jié)果又一致表明2'-OH-BDE-28主要是結(jié)合在HSA的位點(diǎn)I。實(shí)驗(yàn)和計(jì)算機(jī)模擬相互佐證,結(jié)果高度吻合,提高實(shí)驗(yàn)的準(zhǔn)確性,同時(shí)為羥基化多溴二苯醚的毒性機(jī)理的研究提供更可靠的參考信息。

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Combined Simulation and Multispectral Investigation on the Effect of 2'-OH-BDE-28 on the Structure of HSA

Wu Zhiwei1,Yi Zhongsheng1,*,Dong Lu1,Yang Lulu1,Yang Wu1,Zhang Aiqian2
1.Guangxi Colleges and Universities Key Laboratory of Food Safety and Detection,Collaborative Innovation Center for Water Pollution Control and Water Safety in Karst Area,College of Chemistry and Bioengineering,Guilin University of Technology,Guilin 541004,China
2.State Key Laboratory of Environmental Chemistry and Ecotoxicology,Research Center for Eco-environmental Sciences,Chinese A-cademy of Sciences,Beijing 100085,China

13 May 2015 accepted 27 July 2015

In this paper,the interaction mechanism between 2'-hydroxy-2,4,4'-tribromodiphenyl ethers(2'-OHBDE-28)and human serum albumin(HSA)was investigated by combining molecular docking,molecular dynamics simulation(MD)and multispectral techniques.The results of MD simulations indicate that the 2'-OH-BDE-28 induced increase in the interior hydrophobicity of HAS occurred and the molecular structure became loose and expansive,and finally the secondary structure of HSA changed.The results of circular dichroismspectra(CD)arehighly consistent with the results of MD simulations.Moreover,the results of fluorescence spectroscopy experiment also show that the intrinsic fluorescence of HSA was quenched by 2'-OH-BDE-28 through static quenching and non-radiation energy transfer.Molecular docking results indicate that 2'-OH-BDE-28 coordinated with the site I of HAS through hydrophobic forces and hydrogen bonds.Hence,a similar conclusion was obtained from the thermodynamics parameters and competitive experiment to verify the results from molecular docking.Excellent agreement was obtained between computer simulations and multispectral experiments to study the mechanism on the interaction between 2'-hydroxy-2,4,4'-tribromodiphenyl ethers and human serum albumin.

2'-hydroxy-2,4,4'-tribromodiphenyl ethers;human serum albumin;molecular modeling;multispectral analysis

2015-05-13 錄用日期:2015-07-27

1673-5897(2016)1-159-08

X171.5

A

10.7524/AJE.1673-5897.20150513001

伍智蔚,易忠勝,董露,等.模擬和光譜相互驗(yàn)證2'-OH-BDE-28對(duì)HSA構(gòu)象的影響[J].生態(tài)毒理學(xué)報(bào),2016,11(1):159-166

Wu Z W,Yi Z S,Dong L,et al.Combined simulation and multispectral investigation on the effect of 2'-OH-BDE-28 on the structure of HSA[J].Asian Journal of Ecotoxicology,2016,11(1):159-166(in Chinese)

國(guó)家自然科學(xué)基金(21267008,21167006)；廣西自然科學(xué)基金(2013GXNSFAA019034)；廣西高等學(xué)校高水平創(chuàng)新團(tuán)隊(duì)及卓越學(xué)者計(jì)劃項(xiàng)目(桂教人〔2014〕49號(hào))

伍智蔚(1990-),男,碩士,研究方向?yàn)榛瘜W(xué)計(jì)量學(xué)和環(huán)境毒理學(xué),E-mail:wuzwgx@163.com

),E-mail:yzs@glut.edu.cn

簡(jiǎn)介:易忠勝(1970-)，男，博士，教授，研究方向?yàn)榛瘜W(xué)計(jì)量學(xué)和環(huán)境毒理學(xué)。